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Abstract

Background

Approximately 40 species of Sphaeromyxa have been described, all of which are coelozoic parasites from gall bladders of marine
fish. They are unique amongst the myxosporeans as they have polar filaments that are
flat and folded instead of being tubular and spirally wound. This unusual feature
was used as a subordinal character to erect the suborder Sphaeromyxina, which contains
one family, the Sphaeromyxidae, and a single genus Sphaeromyxa.

Methods

In the present study, we examine eelpout from the genus Lycodes from Iceland for the presence of myxosporean parasites in the gall bladder and perform
morphological and DNA studies.

Results

A novel myxosporean, Sphaeromyxa lycodi n. sp., was identified in the gall bladders of five of the six species of Lycodes examined, with a prevalence ranging from 29 - 100%. The coelozoic plasmodia are large,
polysporous and contain disporic pansporoblasts and mature spores which are arcuate.
The pyriform polar capsules encase long and irregularly folded ribbon-like polar filaments.
Each spore valve has two distinct ends and an almost 180° twist along the relatively
indistinct suture line. The single sporoplasm is granular with two nuclei. Sphaeromyxa lycodi is phylogenetically related to other arcuate sphaeromyxids and is reproducibly placed
with all known sphaeromyxids and forms part of a robustly supported clade of numerous
myxosporean genera which infect the hepatic biliary systems of a wide range of hosts.

Conclusions

Sphaeromyxa lycodi is a common gall bladder myxosporean in eelpout of the genus Lycodes from Northern Iceland. It has characteristics typical of the genus and develops arcuate
spores. Molecular phylogenetic analyses confirm that sphaeromyxids form a monophyletic
group, subdivided into straight and arcuate spore forms, within the hepatic biliary
clade that infect a wide range of freshwater associated animals. The ancestral spore
form for the hepatic biliary clade was probably a Chloromyxum morphotype; however, sphaeromyxids have more recently evolved from an ancestor with
a spindle-shaped Myxidium spore form. We recommend that the suborder Sphaeromyxina is suppressed; however,
we retain the family Sphaeromyxidae and place it in the suborder Variisporina.

Keywords:

Background

Myxosporeans are common parasites of fish and have a two-host lifecycle involving
an invertebrate that is generally an annelid worm. The vertebrate host is typically
a fish but other aquatic-associated vertebrates such as turtles, waterfowl and amphibians
as well as terrestrial insectivorous mammals are also reported as hosts [1-5]. There are approximately 40 species described from the genus Sphaeromyxa Thélohan 1892, all of which are coelozoic parasites in gall bladders of marine fish
and form characteristic large flat plasmodia. Although not usually associated with
serious pathology, some may cause blockages of bile ducts which results in bile accumulation
and liver inflammation [6]. Species of this genus are unusual in that they do not have a typical tube-like polar
filament that is spirally wound in the polar capsule. Rather, it is flat in section,
broad at the base, gradually tapering along its length and is folded upon itself several
times in the polar capsule. Lom and Noble [7] proposed this unusual feature as a new subordinal character and erected the suborder
Sphaeromyxina Lom et Noble, 1984 to include a single new family Sphaeromyxidae Lom
et Noble, 1984. In Thélohan’s original description of the genus, Sphaeromyxa, he considered it to be a member of the family Myxidiidae Thelohan 1892. DNA sequence
data for sphaeromyxids are somewhat limited, with information available for only 5
species. However, currently they are one of the few monophyletic myxosporean taxa
[8] and unusually group with a range of other myxosporean genera that infect the gall
bladders of freshwater hosts [9].

There are few reports of myxosporeans from eelpout (Zoarcidae). The type species of
the genus Shulmania, S. ovale, was described from the urinary bladder of Lycodes esmarkii from the Canadian Atlantic [10]. In the Pacific, Myxidium melanostigmum was described from the gall bladder of the eelpout Melanostigma pammelas a deepwater fish off the Californian coast [11] and Myxobolus aeglefini was found in the skeletal muscle of porous-head eelpout Allolepis hollandi from the Sea of Japan [12].

In the present study, we examine eelpout, from the genus Lycodes, from Iceland for the presence of myxosporean parasites in the gall bladder.

Figure 1.Sampling area of Lycodes spp. north of Iceland (shaded area) demarcated by three coordinates.

Fresh material - spore measurements

Thawed fish were dissected, their gall bladder removed and a drop of its contents
put on a microscopic slide and screened for the presence of myxosporean infections
at a magnifications of 200× - 400×. Initially two species of fish were chosen, L. reticulatus and L. eudipleurostictus, and descriptions and measurements of spores were taken following the guidelines
of Lom and Arthur [13]. Fresh spores were measured and photographed using bright field and Nomarski illumination
at magnification up to 1250×. All other fish species were checked for the presence
of myxosporeans and samples taken for DNA analyses.

Histology

Gall bladders from two infected fish, one L. reticulatus and one L. eudipleurostictus, were fixed in 10% buffered formalin, embedded in paraffin wax, sectioned (4 μm),
stained with Giemsa and Haematoxylin and Eosin and prepared for histological examination
according to routine protocols. In addition, air dried smears from infected gall bladders
were fixed in methanol and stained with Giemsa and Haematoxylin and Eosin.

SEM methods

The contents of an infected gall bladder from each species (L. reticulatus and L. eudipleurostictus) of host fish were fixed in 2.5% glutaraldehyde for 4 hrs at 4°C, and then rinsed
four times in 100 mM sodium cacodylate buffer pH 7.2 allowing the spores to settle
under gravity between each rinse. The resulting spore suspension was passed through
a 0.4 μm Whatman Cyclopore® track-etched polycarbonate membrane using a syringe and
filter clamp. The membrane was then post-fixed in 1% osmium tetroxide in 100 mM sodium
cacodylate buffer pH 7.2 for 2hrs and taken through an ethanol series of 30%, 60%,
90% and 2 × 100% 30 mins each, transferred into 50% hexamethyldisilazane (HMDS) in
100% ethanol followed by two changes of 100% HMDS each for 45 min. Excess HMDS was
removed and the membranes allowed to air dry overnight. The membranes were then mounted
onto aluminium stubs, earthed with silver dag paint and sputter-coated with gold.
Samples were viewed with a Jeol JSM 6460 LV SEM instrument.

DNA analysis

Gall bladder contents from three infected fish from, L. reticulatus and L. eudipleurostictus, were used in initial DNA extractions and to obtain the majority (18e-18gM) of the
small subunit ribosomal DNA (SSU rDNA) sequence. Total DNA was extracted using a GeneMATRIX
kit (EURx Poland) following the tissue protocol. Parasite SSU rDNA was amplified using
the myxosporean PCR primers and methodology described by Freeman et al.[14] and the additional primers set 390f 5’agagggagcctgagaaacg 3’ and 1830r 5’ tctaagggcatcacagacctg
3’ using the same PCR conditions. DNA samples from additional infected fish, 2 per
species, L. gracilis, L. pallidus and L. seminudus, were extracted as above and amplified using primers designed to be specific for
sphaeromyxid taxa, Sphy-F 5’gaaaggctcagtatatcag 3’ and Sphy-R 5’ tattcaaggcacgyyatgc
3’ which amplify a 744 base pair region of the SSU rDNA that includes the phylogenetically
informative V4 region. PCR conditions were the same as those described above. All
PCRs were completed in triplicate and PCR products of the expected sizes were recovered
using a GeneMATRIX PCR products extraction kit (EURx Poland).

Sequencing reactions were performed using BigDyeTM Terminator Cycle Sequencing chemistry
utilising the same oligonucleotide primers that were used for the original PCRs. DNA
sequencing was performed in both forward and reverse directions for all PCR products
and nucleotide BLAST searches performed for each sequence read to confirm a myxosporean
origin [15]. The contiguous sequence was obtained manually using CLUSTAL_X and BioEdit [16,17]. CLUSTAL X was used for the initial sequence alignments of 54 myxosporean taxa, with
the settings for gap opening/extension penalties being adjusted manually to achieve
optimum alignments. The final alignment was manually edited using the BioEdit sequence
alignment editor and contained 2619 characters of which 1358 were informative sites.

Phylogenetic analyses were performed using the maximum likelihood methodology in PhyML
[18] with the general time-reversible substitution model selected and 100 bootstrap repeats.
Maximum parsimony in PAUP*4.0 beta10 [19] using a heuristic search with random taxa addition (10 replications), the ACCTRAN-option,
and the TBR swapping algorithm with gaps treated as missing data and branch supports
obtained with 1000 bootstrap replicates. Bayesian inference (BI) analysis using MrBayes
v. 3.2.1 [20]. The BI analysis models of nucleotide substitution were first evaluated for the alignment
using MrModeltest v. 2.2 [21]. The most parameter-rich evolutionary model based on the AIC was the general time-reversible,
GTR+I+G model of evolution. Therefore, the settings used for the analysis were nst
= 6, with the gamma- distributed rate variation across sites and a proportion of invariable
sites (rates = invgamma). The priors on state frequency were left at the default setting
(Prset statefreqpr = dirichlet (1, 1, 1, 1)). Posterior probability distributions
were generated using the Markov Chain Monte Carlo (MCMC) method with four chains being
run simultaneously for 1000,000 generations. Burn in was set at 2500 and trees were
sampled every 100 generations making a total of 7500 trees used to compile the majority
rule consensus trees.

S. lycodi

In accordance with section 8.6 of the ICZN's International Code of Zoological Nomenclature,
details of the new species have been submitted to ZooBank with the Life Science Identifier
(LSID) zoobank.org:pub:B7DFC3E9-5F5A-4239-B811-4C435DAA5423.

Description of Sphaeromyxa lycodi n. sp

The plasmodia are coelozoic, i.e. floating freely in the bile of the gall bladder
and commonly occupying significant parts of the gall bladder’s volume, causing opacity
in some cases. They are polymorphic, long and slender with irregular and long pseudopodial
projections (Figure 2A). They are wrapped around themselves as well as neighbouring plasmodia; similar
to rivets of tangled yarn threads. Estimating their exact length is problematic but
the longest unbroken plasmodium detected in histological sections was approximately
10 mm long. They are polysporous and packed with numerous mature spores and developing
sporoblasts (Figures 2B, C). They form disporic pansporoblasts. Most commonly the ectoplasm is composed
of a narrow (1.0 - 1.5 μm) compact outer layer and a thicker (5 - 7 μm) and triple
eosinophilic inner layer; a finely granular layer enclosed with radially striated
layers (Figure 2D). Occasionally, plasmodia with ectoplasm having spherical bodies and prominent villar
projections were detected (Figure 2E). The endoplasm is vacuolated and loosely connected. In frontal view the spore body
is arcuate and tapers towards its rounded ends. In sutural view the spores are slightly
sigmoid, tapering somewhat towards the blunt ends. The sporoplasm is granular with
a pair of ovoid and centrally located nuclei parallel along the shorter axis. The
pyriform polar capsules encase long and irregularly folded polar filaments. When extruded,
the filaments appear flat along their entire length, broad where they exit the spore
valve and gradually taper along their length (Figures 3A, B and 4A, B).

The suture line is relatively indistinct and similar in appearance to the valve striations,
of which there are 6 – 7 on each spore valve. Each valve has one bulbous / rounded
end that houses the polar capsule with the other end more spoon-like to receive the
polar capsule from the opposing rounded valve end. The valves have an almost 180°
twist along the suture length so that like ends appear to be in the same plane. Striations
are present that start from the rounded end and extend down the valve, parallel to
the suture, but not along the entire length. There is a short terminal striation on
the rounded ends (Figures 5A, B and 6A, B).

Figure 5.Scanning electron micrographs of Sphaeromyxa lycodi n. sp. Spores have an indistinct suture line (white arrows). Each valve has one rounded/bulbous end that supports the polar capsule and the other
more spoon-like to receive the polar capsule from the opposing valve. The valves have
an almost 180° twist along the suture length so that like ends appear to be in the
same plane. Striations are present that start from the rounded/bulbous end extending
along the valve but not for the entire length. There is a terminal short striation
on the rounded/bulbous ends (white asterisk).

Figure 6.(A) and (B) Line drawings showing the frontal view of the two valves of S. lycodi n. sp. separated. Each valve has two different ends; a round/bulbous shaped end (Rbe) and a spoon/cup
shaped end (Sce). The valves have an approximately 180° twist and consequently analogous
ends of the two valves lie in the same plane. In frontal view most of the one valve's
body is visible (A) but only the ends of the opposing valve (B). (C) The suture of the valve ends. The Rbe type appears to sit inside the Sce end and
supports the polar capsule. Scale bars = 2 μm.

DNA analysis

The same myxosporean SSU rDNA sequence was obtained from both L. reticulatus and L. eudipleurostictus, with a contiguous sequence of 1983 bp submitted to Genbank under the accession number
KC524734. Blast searches revealed the closest match in the databases to be Sphaeromyxa kenti and isolates of Sphaeromyxa hellandi, with a 94% and 91% identity respectively. Shorter sequenced regions of the SSU rDNA
from L. gracilis, L. pallidus and L. seminudus all had identical sequence reads to the two type species with longer reads.

Phylogenetic analyses using three methodologies produced congruent tree topologies
with respect to the positioning and members of the major clades (Figure 7). Sphaeromyxa lycodi was reproducibly placed with other sphaeromyxid taxa in all analyses and formed part
of a robustly supported clade of 19 taxa that are found infecting the hepatic biliary
systems of a wide range of hosts (Figure 7). Although the hepatic biliary clade was strongly supported in all analyses, the
relative positions of the 19 taxa varied depending on the phylogenetic methodology
used (Figures 8A, B). Nevertheless, the sphaeromyxids were always robustly placed in a monophyletic
group and were most closely related to Myxidium coryphaenoideum in all analyses. In both maximum likelihood and maximum parsimony topologies M. coryphaenoideum was the basal taxon for the hepatic biliary clade (Figures 7 and 8B), however, in the Bayesian analysis M. coryphaenoideum, together with the sphaeromyxids, formed a sister clade to one containing Myxidium anatidum and Cystodiscus spp. infecting waterfowl and amphibians respectively (Figures 8A). Myxidium hardella and M. chelonarum, from freshwater turtles, consistently grouped together in all analyses and formed
as a sister clade to the fish-infecting species. However, the position of Myxidium scripta, also from freshwater turtles, in the group was not consistent and was poorly supported
in all tree topologies and did not group with other species infecting turtles. Soricimyxum fegati isolated from the liver of the common shrew formed a consistent clade in all analyses
with Chloromyxum trijugum infecting sunfish gall bladder and formed as a sister clade to the fish-infecting
group (Figures 7 and 8A, B).

Figure 7.Maximum likelihood topology based on dataset of 54 aligned myxosporean SSU rDNA sequences,
generated using the general time reversible model of nucleotide substitution in PhyML. Thick branches terminate in a node that received full support from three independent
phylogenetic methodologies, numbers at the nodes refer to bootstrap support values
for maximum likelihood (100 samplings), Bayesian posterior probability support and
percentage bootstrap support for maximum parsimony (1000 samplings), (ns) indicates
an unsupported node or one with a support value below 50. The light red shaded box
represents a well-supported clade of myxosporeans that infect the hepatic biliary
systems of a wide range of host organisms, the number at the nodes in this clade refer
to maximum likelihood support values (see Figure 8 for Bayesian and maximum parsimony topologies for this clade). The darker red shaded
box within the hepatic biliary clade contains Sphaeromyxa lycodi and other sphaeromyxid taxa. The shaded areas bordered by a bold dashed line represent
taxa from the suborder Variisporina, with the exception of the Multivalvulida sequences
from the marine teleost group (blue box).The light orange shaded area, bordered by
a solid line contains taxa from the suborder Platysporina. All myxosporean sequences
were taken from fish hosts unless specified with symbols after the specific names:
representing turtle, shrew, waterfowl or amphibian hosts.The accession numbers of
all sequences used in this analysis are listed in additional file 1.

Figure 8.Part of the phylogenetic trees for Bayesian analysis (A) and maximum parsimony analysis
(B) for the nineteen myxosporean taxa that form the hepatic biliary clade; taken from
trees generated using the same alignment of 54 taxa used in Figure7. Thick branches represent a support value of >95 and (ns) indicates nodes with a support
of <50. Sphaeromyxa lycodi is strongly supported in a clade with other sphaeromyxid taxa and has Myxidium coryphaenoideum as the closest known relative in both trees, but receiving very strong support in
the Bayesian analysis. Shaded boxes represent clades that were recovered in all analyses.

A more focused analysis of all known sphaeromyxid SSU rDNA sequences, including those
with short sequence reads, revealed a robust group divided into strongly supported
sub-clades representing those with straight spores or those with curved ones (Figure 9). Sphaeromyxa lycodi forms a clade with S. kenti, in the group with curved spores with isolates of S. hellandi.

Figure 9.Maximum likelihood phylogeny based on 11 SSU rDNA sequences (2033 characters) of sphaeromyxids
and related taxa.Sphaeromyxa lycodi forms a robust clade with S. kenti, which is a well-supported sister clade to the S. hellandi group. The sphaeromyxids form two robustly supported groups from node A (arrowed);
one clade contains taxa with straight spores (blue box) and the other contains those
with curved spores (green box). Figures at the nodes represent percentage bootstrap
support values from 1000 samplings. Cystodiscus melleni is used as the outgroup and to root the tree.

The only sequence from non-fish host that grouped outside the hepatic biliary clade
was Chloromyxum careni isolated from the kidney of the Malayan horned frog Megophrys nasuta[22].

Discussion

To date approximately 40 Sphaeromyxa species have been reported, all of which are parasitic in the hepatic biliary systems
of marine fishes, typically found in the gall bladder. On the basis of the morphological
features of mature spores, they have been divided into two main groups; having either
arcuate or straight spores [23], and DNA analysis in the present paper, based on available Sphaeromyxa sequences, supports this grouping (Figure 9). Sphaeromyxids have been shown to have unusually low host specificity [24], a characteristic also demonstrated in this study with 5 of the 6 Lycodes spp. as hosts. As DNA data for the group is limited (six species now have SSU rDNA
data, Figure 9), most descriptions of sphaeromyxids have been based exclusively on morphological
characteristics, and therefore, it is important to demonstrate that potential new
species are novel. The arcuate species S. hellandi and S. kenti[8,25] are the most phylogenetically related to S. lycodi n. sp., but are too distant to be the considered conspecific (Figure 9). Five other known arcuate species, S. arcuata, S. curvula, S. sabrazesi, S. elegini and S. noblei, show some resemblance to S. lycodi with regard to spore size. However, when compared they are all quite different with
respect to one or more features (Table 1). Firstly, all these species have very different geographic distributions with one
reported from the Mediterranean, one from Australian waters, one from Japan and Barents
Sea and one from the South Atlantic [23,24,26]. With regard to morphology, S. arcuata and S. curvula, have considerably more slender polar capsules than S. lycodi. Furthermore, S. arcuata is generally slightly longer while S. curvula is slightly shorter than S. lycodi[23]. Both the spore body and the polar capsules of S. sabrazesi are significantly narrower in addition to the polar filaments being only half the
length of those from S. lycodi[23]. Sphaeromyxa elegini is different in having a very small (10 × 18 μm) disporous plasmodia but also a differently
arranged nuclei [26]. Finally, S. noblei has a leaf-like plasmodia and differently arranged nuclei [24]. Scanning electron microscopy of spores of S. lycodi has allowed us to visualise the shape and arrangement of the two valves (Figures 5 and 6), which appear to be somewhat similar to those in the phylogenetically related species
S. kenti. This valvular arrangement, one rounded end and one cupped end, may be a common feature
in arcuate spore forms but SEM data is limited for the group.

Table 1.Comparison of S. lycodi with other arcuate Sphaeromyxa species which overlap with regard to spore length[8,23,25,26]

Although L. reticulatus and L. eudipleurostictus, the type hosts for S. lycodi, occupy the same genus, they are readily phylogenetically distinguished from each
other using both morphological (tail length) and DNA sequence data [27]. Lycodes reticulatus forms part of the monophyletic short tail group that is also supported in multiple
gene phylogenies, whilst L. eudipleurostictus is part of the sister clade of long-tailed species [27]. We have demonstrated that fish from both major groups of Lycodes are host to S. lycodi, with the exception of L. esmarkii. Lycodes esmarkii is a member of the clade of long-tailed species, and hence may also be a host for
S. lycodi, but was not detected in this study. It is also possible that other related fish
genera could be susceptible to infection with S. lycodi. Indeed, other sphaeromyxids such as S. hellandi and the type species S. balbianii are known from multiple fish hosts, often distantly related [24], indicating that sphaeromyxids are not routinely host specific and susceptibility
to infection may be due to other factors. The prevalence of infection in adult Lycodes spp. was high in most cases, and apart from opacity in some gall bladders no pathology
was apparent, suggesting that S. lycodi is not pathogenic to the host.

Myxosporeans tend to have a characteristic size of SSU rDNA depending whether they
are marine or freshwater species and typically form reliable freshwater and marine
clades in phylogenetic analyses [9]. The length of the SSU rDNA sequence for S. lycodi is comparable to that of other sphaeromyxids, which are more similar to freshwater
myxosporeans than marine species [9]. Indeed, in all of our phylogenetic analyses, the sphaeromyxids are robustly supported
in a discrete clade of myxosporeans that infect the hepatic biliary systems of numerous
freshwater associated hosts, including fishes, turtles, amphibians, waterfowl and
terrestrial insectivorous mammals. However, sphaeromyxids are all described from marine
fish, including those from deep water environments such as the Lycodes in this study. Our phylogenetic analyses also demonstrate that sphaeromyxids form
a well-supported monophyletic group within this freshwater clade and share a common
ancestor with Myxidium coryphaenoideum. Myxidium coryphaenoideum and morphologically similar species M. melanostigum, M. melanocetum and M. bajacalifornium all share numerous characteristics with sphaeromyxids. They are elongate spindle-like
myxosporeans with two nuclei; all are known to develop large polysporous plasmodia
in the gall bladders of deep sea fish, some with ‘heavy’ polar filaments [11]. Myxidium coryphaenoideum is also known to exhibit low host specificity and have an atypical ‘rough’ polar
filament [28,29]. These similarities to Sphaeromyxa spp. support this type of ancestral Myxidium as the correct morphotype for the sphaeromyxids. Fiala [9] supplied the SSU rDNA sequence for M. coryphaenoideum and in his phylogenetic analyses he also found it grouped basally to sequences for
sphaeromyxids and formed part of a clade of myxosporeans infecting the gall bladders
of freshwater fishes. Fiala [9] concluded that sphaeromyxids are closely related to Myxidium species, M. coryphaenoideum being the closest species and suggested that the common ancestor of marine Sphaeromyxa spp. was a freshwater myxosporean with Myxidium-shaped spores. We agree that the evidence, both morphological and molecular, is highly
indicative that all sphaeromyxids evolved from a common ancestor with an elongate
spindle form, similar to that of M. coryphaenoideum and the DNA data is supportive of a freshwater origin. However, what is less clear
is whether this spore form is the ancestral morphotype for the well-supported hepatic
biliary clade (Figures 7 and 8). It has been well reported that Myxidium and Zschokkella-shaped spores are polyphyletically distributed within myxosporean systematics and
hence are assumed to have evolved on numerous occasions throughout myxosporean evolution
[9,30,31]. It may be possible that all myxosporean spore forms are as plastic as Myxidium over evolutionary time, or it may be that some forms evolve at slower rates and are
more likely to be true ancestral morphotypes for clades such as the hepatic biliary
group. The majority of known taxa in the hepatic biliary clade (Myxidium, Zschokkella, Sphaeromyxa, Cystodiscus and Soricimyxum) could have, and likely did, all evolve from an immediate Myxidium morphotype ancestry. However, the unambiguous inclusion of Chloromyxum trijugum in the group makes it unlikely that the ancestral morphotype for the hepatic biliary
clade was Myxidium-like. A study of the history of character evolution in myxozoans also indicates that
the Chloromyxum spore morphotype was more stable during the evolution of the myxosporeans and was
responsible for the radiation of freshwater myxosporeans after separation from the
marine Chloromyxum leydigi group [31] that infects various elasmobranchs (Figure 7). Therefore, we consider it more likely that the ancestral spore form for the hepatic
biliary clade was a Chloromyxum morphotype.

Myxosporeans from the hepatic biliary clade infecting gall bladders of invasive amphibians,
such as the cane toad (Bufo marinus) in Australia, are known to spread to numerous endemic species [32,33], again indicating the very low host specificity found in this group of myxosporeans.
The spread of wildlife pathogens into new geographical ranges or populations is a
conservation concern for endangered species of which amphibian decline is one of the
most dramatic examples.

Myxosporeans that infect certain organs or tissues have been shown to reproducibly
cluster together in molecular phylogenetic analyses [9,34] and all taxa in the hepatic biliary group are found infecting the gall bladder, bile
ducts or liver of their hosts. However, Myxidium scripta and M. hardella, infecting freshwater turtles, are also reported from renal tubules as well as from
the bile ducts and gall bladder [35,36]. This may be due to these reports being from systemic infections, as in both cases
severe pathologies and mortalities had occurred, and it is possible that the hepatic
biliary system is the initial site of infection with other organs only becoming infected
during the advanced stages of infection. However, it may also be possible that turtle
myxosporeans in this clade are an exception to this pattern. Currently only a single
taxon, Chloromyxum careni, isolated from kidney tissues alone, from a non-fish host groups outside the hepatic
biliary clade. In our analyses it forms a moderately supported group with other species
of Chloromyxum that infect the gall bladders of freshwater fishes (Figure 7). However, in other analyses that are more focused on the phylogenetic relationships
amongst Chloromyxum spp., its position is unresolved and it forms a solitary branch between a clade of
urinary bladder infecting species and the Myxidium lieberkuehni clade, both of which contain Chloromyxum taxa [37]. It is likely that, when more molecular data exist for myxosporean taxa from the
renal systems of amphibians, they will form a clade with C. careni reinforcing the potential importance of the Chloromyxum morphotype as ancestral forms to some of the currently recognised clades in myxosporean
systematics.

The sphaeromyxids are currently classified in a separate suborder, the Sphaeromyxina
Lom et Noble, 1984, due to the presence of the unique ribbon-like polar filament they
all possess. Sphaeromyxids do form a monophyletic clade in this and other phylogenetic
studies [8,9], including multiple gene analyses [38] suggesting that this feature is unique amongst the myxosporeans and was derived from
an ancestor common to all known sphaeromyxids. However, their assignment to a separate
suborder is no longer justified as they are robustly located within taxa from the
suborder Variisporina Lom et Noble, 1984, in phylogenetic analyses and have clearly
evolved from a common ancestor with an elongate Myxidium form, similar to that of M. coryphaenoideum. Other recent molecular studies on sphaeromyxids [9,39] and Lom and Dyková’s synopsis of myxozoan genera [40] support these findings; therefore, we recommend that the suborder Sphaeromyxina is
no longer retained. However, due to the polyphyletic nature of Myxidium in myxosporean systematics, and the placement of the type species, M. lieberkuehni, in a different clade to the sphaeromyxids (Figure 7), we retain the family Sphaeromyxidae and place it in the suborder Variisporina Lom
et Noble, 1984.

Taxanomic summary

Phylum: Cnidaria Hatschek, 1888

Unranked subphylum: Myxozoa Grassé 1970

Class: Myxosporea Bütschli, 1881

Order: Bivalvulida Schulman, 1959

Suborder: Variisporina Lom et Noble, 1984

Family: Sphaeromyxidae Lom et Noble, 1984

Genus: Sphaeromyxa Thélohan, 1892

Amended description for genus Sphaeromyxa

Polar filament is not tube-like as in other Myxosporea, being flat with a broad base
that gradually tapers to the end. In the polar capsule it is irregularly folded several
times instead of being spirally wound. Polysporous plasmodia containing disporic pansporoblasts
are coelozoic in the gall bladder and bile ducts of marine fishes, and may be several
mm in length or diameter. Spore elongated, sometimes slightly curved or arcuate; the
two polar capsules lie in its opposite, tapering and truncate ends. Spores open at
the level of the straight or curved suture line, bisecting the spore and connecting
both its ends. Shell valves smooth or ridged. One binucleate sporoplasm. Marked pathology
may result in forms that infect bile ducts.

Main amendments include: polar filament no longer described as short, and pathology
may result from infection.

Specific diagnosis of Sphaeromyxum lycodi n. sp

Large polysporous plasmodia, up to 10 mm in length, containing disporic pansporoblasts,
are coelozoic in the gall bladder of Lycodes spp. In frontal view the spore body is arcuate and tapers towards its rounded ends.
In sutural view the spores are slightly sigmoid, tapering somewhat towards the blunt
ends. The suture line is relatively indistinct and similar in appearance to the valve
striations. Each valve has one bulbous / rounded end that supports the polar capsule
with the other end more spoon-like to receive the polar capsule from the opposing
rounded valve end. The valves have an almost 180° twist along the suture length so
that like ends appear to be in the same plane; there are 6 – 7 striations present
on each valve. The pyriform polar capsules encase relatively long and irregularly
folded polar filaments. When extruded, the filaments appear flat along their entire
length, broad where they exit the spore valve and gradually taper along their length.
The sporoplasm is granular with a pair of ovoid and centrally located nuclei.

A SSU rDNA sequence was submitted to Genbank under the accession number KC524734

Conclusions

Sphaeromyxa lycodi n. sp. is a common gall bladder myxosporean in numerous eelpout of the genus Lycodes from Northern Iceland. It has characteristics typical of the genus and forms arcuate
spores. The spore valves have one rounded end that supports the polar capsule and
one spoon-like end to receive the polar capsule from the opposing rounded valve. Molecular
phylogenetic analyses confirm that sphaeromyxids form a monophyletic group, subdivided
into straight and arcuate spore forms, within the hepatic biliary clade that infect
a wide range of freshwater associated animals. The ancestral spore form for the hepatic
biliary clade was probably a Chloromyxum morphotype; however, sphaeromyxids have more recently evolved from a Myxidium ancestor with a spindle-shaped spore. We recommend that the suborder Sphaeromyxina
is suppressed; however, we retain the family Sphaeromyxidae and place it in the suborder
Variisporina.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

ÁK and MF dissected the fish and isolated the myxosporeans. ÁK performed the morphological
and histological studies. MF carried out the DNA analyses and the SEM. ÁK and MF jointly
wrote the manuscript. Both authors approved the final version of the manuscript.

Acknowledgements

We would like to thank the staff at the Marine Research Institute in Iceland and the
crew of the vessel for assistance with sampling the fish. Funding for the molecular
study was provided by a University of Malaya Research Grant (UMRG) No: RG201-12SUS
and partial funding for publication costs was provided by the University of Malaya
RU fund.

Hartigan A, Fiala I, Dyková I, Rose K, Phalen DN, Šlapeta J: New species of Myxosporea from frogs and resurrection of the genus Cystodiscus Lutz, 1889 for species with myxospores in gallbladders of amphibians.